Endostatin protein and fragments thereof

ABSTRACT

The present invention provides anti-angiogenic compositions, and more particularly provides endostatin protein fragments. The compositions of the present invention may be used for the treatment of angiogenesis-dependent diseases such as cancer.

CROSS REFERENCE TO PRIOR RELATED CASES

This application is a continuation application of U.S. patentapplication Ser. No. 09/154,302, filed Sep. 16, 1998, now U.S. Pat. No.6,630,448 which is a divisional application of U.S. patent applicationSer. No. 08/740,168, filed Oct. 22, 1996 and issued as U.S. Pat. No.5,854,205, which claims priority to provisional application Serial No.60/005,835 filed October 23, 1995; provisional application Serial No.60/023,070 filed Aug. 2, 1996; and provisional application Serial No.60/026,263 filed Sep. 17, 1996. Each of the above-referencedapplications is incorporated herein in its entirety.

This invention may have been made in part by funds from NIH grantsRO1-CA64481 and PO1-CA45548. The U.S. government may have certain rightsin this invention.

TECHNICAL FIELD

This application relates to a novel inhibitor of angiogenesis useful fortreating angiogenesis-related diseases, such as angiogenesis-dependentcancer. The invention further relates to a novel composition and methodfor curing angiogenesis-dependent cancer. In addition, the presentinvention relates to diagnostic assays and kits for endostatinmeasurement, to histochemical kits for localization of endostatin, tomolecular probes to monitor endostatin biosynthesis, to antibodies thatare specific for the endostatin, to the development of peptide agonistsand antagonists to the endostatin receptor, and to cytotoxic agentslinked to endostatin peptides.

BACKGROUND OF THE INVENTION

Several lines of direct evidence now suggest that angiogenesis isessential for the growth and persistence of solid tumors and theirmetastases (Folkman, 1989; Hori et al., 1991; Kim et al., 1993; Millaueret al., 1994). To stimulate angiogenesis, tumors upregulate theirproduction of a variety of angiogenic factors, including the fibroblastgrowth factors (FGF and BFGF) (Kandel et al., 1991) and vascularendothelial cell growth factor/vascular permeability factor (VEGF/VPF).However, many malignant tumors also generate inhibitors of angiogenesis,including angiostatin and thrombospondin (Chen et al., 1995; Good etal., 1990; O'Reilly et al., 1994). It is postulated that the angiogenicphenotype is the result of a net balance between these positive andnegative regulators of neovascularization (Good et al., 1990; O'Reillyet al., 1994; Parangi et al., 1996; Rastinejad et al., 1989). Severalother endogenous inhibitors of angiogenesis have been identified,although not all are associated with the presence of a tumor. Theseinclude, platelet factor 4 (Gupta et al., 1995; Maione et al., 1990),interferon-alpha, interferon-inducible protein 10 (Angiolillo et al.,1995; Strieter et al., 1995), which is induced by interleukin-12 and/orinterferon-gamma (Voest et al., 1995), gro-beta (Cao et al., 1995), andthe 16 kDa N-terminal fragment of prolactin (Clapp et al., 1993). Theonly known angiogenesis inhibitor which specifically inhibitsendothelial cell proliferation is angiostatin (O'Reilly et al. 1994).

Angiostatin is an approximately 38 kiloDalton (kDa) specific inhibitorof endothelial cell proliferation. Angiostatin is an internal fragmentof plasminogen containing at least three of the five kringles ofplasminogen Angiostatin has been shown to reduce tumor weight and toinhibit metastasis in certain tumor models. (O'Reilly et al., 1994). Asit is used hereinafter, the term “angiostatin” refers to angiostatin asdescribed above; peptide fragments of angiostatin that have endothelialcell proliferation inhibiting activity; and analogs of angiostatin thathave substantial sequence homology (as defined herein) to the amino acidsequence of angiostatin, which have endothelial cell proliferationinhibiting activity.

SUMMARY OF THE INVENTION

The present invention relates to a novel protein inhibitor, and methodfor its use. The protein is a potent and specific inhibitor ofendothelial proliferation and angiogenesis. Systemic therapy with theinhibitor causes a nearly complete suppression of tumor-inducedangiogenesis, and it exhibits strong anti-tumor activity.

The inhibitory protein has a molecular weight of approximately 18,000 toapproximately 20,000 Daltons (18 to 20 kDa) and is capable of inhibitingendothelial cell proliferation in cultured endothelial cells. Theprotein can be further characterized by its preferred N-terminal aminoacid sequence, the first twenty (20) of which are as follows:

His Thr His Gln Asp Phe Gln Pro Val Leu (SEQ ID1   2   3   4   5   6   7   8   9   10 NO:1) His Leu Val Ala Leu Asn ThrPro Leu Ser 11  12  13  14  15  16  17  18  19  20

A preferred endothelial cell proliferation inhibitor of the invention isa protein having the above-described characteristics, and which can beisolated and purified from the murine hemangioendothelioma cell lineEOMA. This inhibitory protein has been named endostatin.

The present invention provides methods and compositions for treatingdiseases and processes mediated by undesired and uncontrolledangiogenesis by administering to a human or animal with the undesiredangiogenesis a composition comprising a substantially purifiedendostatin or endostatin derivative in a dosage sufficient to inhibitangiogenesis. The present invention is particularly useful for treatingor for repressing the growth of tumors. Administration of endostatin toa human or animal with prevascularized metastasized tumors prevents thegrowth or expansion of those tumors.

The present invention also includes diagnostic methods and kits fordetection and measurement of endostatin in biological fluids andtissues, and for localization of endostatin in tissues. The diagnosticmethod and kit can be in any configuration well known to those ofordinary skill in the art. The present invention also includesantibodies specific for the endostatin and antibodies that inhibit thebinding of antibodies specific for the endostatin. These antibodies canbe polyclonal antibodies or monoclonal antibodies. The antibodiesspecific for endostatin can be used in diagnostic kits to detect thepresence and quantity of endostatin which is diagnostic or prognosticfor the occurrence or recurrence of cancer or other diseases mediated byangiogenesis. Antibodies specific for endostatin may also beadministered to a human or animal to passively immunize the human oranimal against endostatin, thereby reducing angiogenic inhibition.

The present invention also includes diagnostic methods and kits fordetecting the presence and quantity of antibodies that bind endostatinin body fluids. The diagnostic method and kit can be in anyconfiguration well known to those of ordinary skill in the art.

The present invention also includes endostatin peptide fragments thatcan be labeled isotopically or with other molecules or proteins for usein the detection and visualization of endostatin binding sites withstate of the art techniques, including, but not limited to, positronemission tomography, autoradiography, flow cytometry, radioreceptorbinding assays, and immunohistochemistry.

These endostatin peptides also act as agonists and antagonists at theendostatin receptor, thereby enhancing or blocking the biologicalactivity of endostatin. Such peptides are used in the isolation of theendostatin receptor.

The present invention also includes endostatin, endostatin fragments,endostatin antisera, or endostatin receptor agonists and antagonistslinked to cytotoxic agents for therapeutic and research applications.

The present invention includes molecular probes for the ribonucleic acidand deoxyribonucleic acid involved in transcription and translation ofendostatin. These molecular probes provide means to detect and measureendostatin biosynthesis in tissues and cells.

A surprising discovery is that various forms of recombinant endostatinprotein can serve as sustained release anti-angiogenesis compounds whenadministered to a tumor-bearing animal. A preferred form of thesustained release compound is un-refolded recombinantly producedendostatin.

Additionally, the present invention encompasses nucleic acid sequencescomprising corresponding nucleotide codons that code for the abovedisclosed amino acid sequence and for endostatin and endothelial cellproliferation inhibiting peptide fragments thereof.

The present invention also relates to methods of using the endostatinprotein and peptide fragments, corresponding nucleic acid sequences, andantibodies that bind specifically to the inhibitor and its peptides, todiagnose endothelial cell-related diseases and disorders.

The invention further encompasses a method for identifying receptorsspecific for endostatin, and the receptor molecules identified andisolated thereby.

The invention also relates to a method for identifying novel enzymescapable of releasing endostatin from collagen type XVIII, and othermolecules containing an endostatin amino acid sequence, and peptidesthereof. Such endostatin producing enzymes are also an aspect of theinvention.

An important medical method is a new form of birth control, wherein aneffective amount of endostatin is administered to a female such thatuterine endometrial vascularization is inhibited and embryo implantationcannot occur, or be sustained.

A particularly important aspect of the present invention is thediscovery of a novel and effective method for treatingangiogenesis-related diseases, particularly angiogenesis-dependentcancer, in patients, and for curing angiogenesis-dependent cancer inpatients. The method unexpectedly provides the medically importantresult of inhibition of tumor growth and reduction of tumor mass. Themethod relates to the co-administration of the endostatin of the presentinvention and another anti-angiogenesis compound, preferrablyangiostatin. Accordingly, the present invention also includesformulations containing endostatin, and optionally angiostatin, whichare effective for treating or curing angiogenesis-dependent cancers.

Accordingly, it is an object of the present invention to provide acomposition comprising an endostatin protein.

It is another object of the present invention to provide a method oftreating diseases and processes that are mediated by angiogenesis.

It is yet another object of the present invention to provide adiagnostic or prognostic method and kit for detecting the presence andamount of endostatin in a body fluid or tissue.

It is yet another object of the present invention to provide a methodand composition for treating diseases and processes that are mediated byangiogenesis including, but not limited to, hemangioma, solid tumors,leukemia, metastasis, telangiectasia psoriasis scleroderma, pyogenicgranuloma, myocardial angiogenesis, plaque neovascularization, corornaycollaterals, cerebral collaterals, arteriovenous malformations, ischemiclimb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma,diabetic retinopathy, retrolental fibroplasia, arthritis, diabeticneovascularization, macular degeneration, wound healing, peptic ulcer,fractures, keloids, vasculogenesis, hematopoiesis, ovulation,menstruation, and placentation.

It is another object of the present invention to provide a compositionfor treating or repressing the growth of a cancer.

It is an object of present invention to provide a method for detectingand quantifying the presence of an antibody specific for an endostatinin a body fluid.

Still another object of the present invention is to provide acomposition consisting of antibodies to endostatin that are selectivefor specific regions of the endostatin molecule.

It is another object of the present invention to provide a method forthe detection or prognosis of cancer.

It is another object of the present invention to provide a compositionfor use in visualizing and quantitating sites of endostatin binding invivo and in vitro.

It is yet another object of the present invention to provide acomposition for use in detection and quantification of endostatinbiosynthesis.

It is yet another object of the present invention to provide a therapyfor cancer that has minimal side effects.

Still another object of the present invention is to provide acomposition comprising endostatin or an endostatin peptide linked to acytotoxic agent for treating or repressing the growth of a cancer.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Inhibition of Capillary Endothelial Cell Proliferation byConditioned Media from EOMA Cells.

Conditioned media collected from confluent EOMA cells or base media wasapplied to bovine capillary endothelial cells with 1 ng/ml bFGF in a 72hour proliferation assay. Endothelial cell proliferation was inhibitedby the EOMA conditioned media. Each bar represents the mean±SEM.

FIG. 2: Purification of an Inhibitor of Endothelial Proliferation fromEOMA Conditioned Media.

Conditioned media collected from confluent EOMA cells was fractionatedon a heparin sepharose column. Endothelial proliferation inhibitingactivity eluted at approximately 0.8M NaCl.

FIG. 3: Purification of an Inhibitor of Endothelial Proliferation by GelFiltration.

Purified inhibitor from heparin sepharose column chromatography wasapplied to a gel filtration column and eluted as a single peak.

FIG. 4: Purification of Inhibitor of Endothelial Cell Proliferation byReversed Phase Column Chromatography.

Inhibitor purified by heparin sepharose and gel filtrationchromatography was applied to a reverse phase column. The inhibitoreluted as a single band from the column at approximately 45% of theacetonitrile.

FIG. 5: N-terminal Amino Acid Sequence of An Inhibitor of EndothelialCell Proliferation.

The N-terminal sequence of the purified inhibitor of endothelial cellproliferation is shown in relation to a schematic diagram of collagentype 18. The N-terminal sequence revealed identity of the inhibitor toan approximately 20 kDa C-terminal fragment (shown in solid shading) forcollagen type XVIII. The open boxes represent the collagenase domains ofcollagen type XVIII.

FIG. 6: Treatment of Lewis Lung Carcinoma With Recombinant MouseEndostatin Inhibitor.

Recombinant inhibitor produced in E. coli was administered to miceseeded with Lewis lung carcinoma that had achieved a tumor volume ofapproximately 150 mm³. The inhibitor was administered at 20 mg/kg/day.Tumor mass regressed to non-detectable levels after approximately 12days of treatment.

FIGS. 7A-C: Systemic Therapy with Recombinant Endostatin Regresses LewisLung Carcinoma Primary Tumors.

FIG. 7A. Mice were implanted subcutaneously on the dorsum with Lewislung carcinoma cells. Systemic therapy with recombinant mouse endostatin(20 mg/kg/day) demonstrated that endostatin inhibitor inhibited tumorsby >99% relative to saline-treated controls. Each point representsmean±SEM for 5 mice. The experiment was repeated with comparableresults.

FIG. 7B. Representative treated and untreated tumor-bearing mice after11 days of systemic therapy with endostatin. Saline-treated mice (right)had rapidly growing red tumors with ulcerated surfaces. Endostatintreated mice (left) had small pale residual tumors (arrow).

FIG. 7C. Residual disease in endostatin treated mice. Three of the fiveendostatin treated mice were sacrificed after 16 days of therapy.Autopsy revealed small white residual tumors at the site of the originalprimary implantation (arrows).

FIG. 8: Treatment of Murine T241 Fibrosarcoma with Recombinant MouseEndostatin from E. coli.

Mice were seated with T241 Fibrosarcoma cells. Control mice were treatedwith saline. Experimental mice were treated with 20 mg/kg/day ofrecombinant mouse Endostatin directed from E. coli.

FIG. 9: Treatment of Murine B16F10 Melanoma with Recombinant MouseEndostatin from E. coli.

Mice were seated with Murine B16F10 melanoma cells. Control animals weretreated with saline. Experimental animals were treated with 20 mg/kg/dayof recombinant mouse Endostatin direct from E. coli.

FIG. 10: Treatment of EOMA Hemangioendothelioma with Recombinant MouseEndostatin from E. coli.

Mice were seated with EOMA hemangioendothelioma cells. Control animalswere treated with saline. Experimental animals were treated with 20mg/kg/day of Recombinant Mouse Endostatin direct from E. coli.

FIG. 11: Treatment of Lewis Lung Carcinoma with Recombinant Mouse orHuman Endostatin direct from E. coli.

Mice were seated with Lewis Lung Carcinoma cells. Control animals weretreated with saline. Experimental animals were treated with RecombinantEndostatin derived from the mouse sequence or Recombinant Endostatindirect from the human sequence, wherein both Endostatin were producedrecombinantly in the E. coli. Mouse Endostatin was administered ateither 20 mg/kg/day or 2.5 mg/kg/day, and Human Endostatin wasadministered at 20 mg/kg/day.

FIGS. 12A-C: Endostatin Results in an Inhibition of Angiogenesis and anIncrease in Apoptosis of Lewis Lung Carcinoma Primary Tumors.

Histological sections of tumors from saline versus endostatin treatedmice implanted with Lewis lung carcinomas were analyzed forproliferation (PCNA), apoptosis (TUNEL), and angiogenesis (vWF). Therewas no significant difference in the proliferative index of tumor cells(FIG. 12A) in treated versus untreated tumors. In contrast, theapoptotic index of the tumor cells (FIG. 12B) increased 8-fold (p<0.001)in the endostatin treated mice. Vessel density (FIG. 12C) was determinedby counting the number of capillary blood vessels per high-power field(HPF) in sections stained with antibodies against vWF. Angiogenesis wasalmost completely suppressed in the residual microscopic tumors of theendostatin treated mice (p<0.001).

FIG. 13: Cycle Dormancy Therapy of Lewis Lung Carcinoma with RecombinantMouse Endostatin From E. coli.

Mice were implanted subcutaneously on the dorsum with Lewis lungcarcinoma cells. Systemic therapy with recombinant mouse inhibitor(endostatin), administered at a dose of 20 mg/kg/day, was begun whentumors were approximately 200 mm³ (1% of body weight). Tumors in themice treated with the endostatin inhibitor rapidly regressed toessentially non-detectable levels after approximately 15 days oftherapy. When treatment was terminated the tumor volume increasedrapidly and was subsequently treatable to the same non-detectable levelsby reinitiation of treatment. The peaks and valleys in the figure showthe cycling effect of inhibition with endostatin.

FIG. 14: Combination Therapy with Recombinant Mouse Angiostatin andEndostatin from E. coli.

Mice were implanted subcutaneously on the dorsum with Lewis lungcarcinoma cells. Systemic therapy with a combination of recombinantmouse endostatin (20 mg/kg/day) and recombinant mouse angiostatin (20mg/kg/day) was begun when tumors were approximately 300 mm³. Tumors inthe mice treated with the combination therapy rapidly regressed toessentially non-detectable level in about 15 days. Importantly, theregressed tumors remained dormant and did not increase in size or massafter treatment was stopped. This is an unexpected result of substantialmedical significance.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have discovered a new class of protein molecules that havethe ability to inhibit endothelial proliferation when added toproliferating endothelial cells in vitro. Accordingly, these proteinmolecules have been functionally defined as endostatins, however, it isto be understood that this functional definition is no way limits thebioactivity of endostatins to inhibition of endothelial cell growth invitro or in vivo. Many other functions of endostatins are likely.

The term “endostatin” refers to a protein that is preferably 18 kDa to20 kDa in size as determined by non-reduced and reduced gelelectrophoresis, respectively. The term endostatin also includesprecursor forms of the 18 kDa to 20 kDa protein. The term endostatinalso includes fragments of the 18 kDa to 20 kDa protein and modifiedproteins and peptides that have a substantially similar amino acidsequence, and which are capable inhibiting proliferation of endothelialcells. For example, silent substitutions of amino acids, wherein thereplacement of an amino acid with a structurally or chemically similaramino acid does not significantly alter the structure, conformation oractivity of the protein, is well known in the art. Such silentsubstitutions are intended to fall within the scope of the appendedclaims.

It will be appreciated that the term “endostatin” includes shortenedproteins or peptides wherein one or more amino acid is removed fromeither or both ends of endostatin, or from an internal region of theprotein, yet the resulting molecule retains endothelial proliferationinhibiting activity. The term “endostatin” also includes lengthenedproteins or peptides wherein one or more amino acid is added to eitheror both ends of endostatin, or to an internal location in the protein,yet the resulting molecule retains endothelial proliferation inhibitingactivity. Such molecules, for example with tyrosine added in the firstposition are useful for labeling such as radioiodination with ¹²⁵iodinefor use in assays. Labelling with other radioisotopes may be useful inproviding a molecular tool for destroying the target cell containingendostatin receptors. Other labeling with molecules such as ricin mayprovide a mechanism for destroying cells with endostatin receptors.

“Substantial sequence homology” means at least approximately 70%homology between amino acid residue sequence in the endostatin analogsequence and that of endostatin, preferably at least approximately 80%homology, more preferably at least approximately 90% homology.

Also included in the definition of the term endostatin are modificationsof the endostatin protein, its subunits and peptide fragments. Suchmodifications include substitutions of naturally occurring amino acidsat specific sites with other molecules, including but not limited tonaturally and non-naturally occurring amino acids. Such substitutionsmay modify the bioactivity of endostatin and produce biological orpharmacological agonists or antagonists. The term endostatin alsoincludes an N terminal fragment of endostatin consisting of the sequenceof the first 20 N terminal amino acids which is shown in SEQ ID NO:1 andis shown in Table 1. This sequence of the first 20 N terminal aminoacids corresponds to a C-terminal fragment of newly identified collagentype XVIII.

Table 1 shows the correspondence of 3 letter and 1 letter designations.

TABLE 1 Amino Acid Residue Abbreviation 1 HIS H 2 THR T 3 HIS H 4 GLN Q5 ASP D 6 PHE F 7 GLN Q 8 PRO P 9 VAL V 10 LEU L 11 HIS H 12 LEU L 13VAL V 14 ALA A 15 LEU L 16 ASN N 17 THR T 18 PRO P 19 LEU L 20 SER S

The N-terminal amino acid sequence of endostatin corresponds to aninternal 20 amino acid peptide fragment found in mouse collagen alpha 1type XVIII starting at amino acid 1105 and ending at amino acid 1124.The N-terminal amino acid sequence of the inhibitor also corresponds toan internal 20 amino acid peptide fragment found in human collagen alpha1 type XVIII starting at amino acid 1132 at amino acid 1151.

Endostatin can be isolated from murine hemangioendothelioma EOMA.Endostatin may be produced from recombinant sources, from geneticallyaltered cells implanted into animals, from tumors, and from cellcultures as well as other sources. It is anticipated that endostatin ismade in cells of the nervous system. Endostatin can be isolated frombody fluids including, but not limited to, serum, urine and ascites, orsynthesized by chemical or biological methods (e.g. cell culture,recombinant gene expression, peptide synthesis, and in vitro enzymaticcatalysis of precursor molecules to yield active endostatin).Recombinant techniques include gene amplification from DNA sources usingthe polymerase chain reaction (PCR), and gene amplification from RNAsources using reverse transcriptase/PCR.

Endostatin specifically and reversibly inhibits endothelial cellproliferation. The inhibitor protein molecules of the invention areuseful as a birth control drug, and for treating angiogenesis-relateddiseases, particularly angiogenesis-dependent cancers and tumors. Theprotein molecules are also useful for curing angiogenesis-dependentcancers and tumors. The unexpected and surprising ability of these novelcompounds to treat and cure angiogenesis-dependent cancers and tumorsanswers a long felt unfulfilled need in the medical arts, and providesan important benefit to mankind.

Important terms that are used herein are defined as follows. “Cancer”means angiogenesis-dependent cancers and tumors, i.e. tumors thatrequire for their growth (expansion in volume and/or mass) an increasein the number and density of the blood vessels supplying them withblood. “Regression” refers to the reduction of tumor mass and size.

The endothelial proliferation inhibiting proteins of the presentinvention can be made by automated protein synthesis methodologies wellknown to one skilled in the art. Alternatively, endothelialproliferation inhibiting proteins, or endostatins, of the presentinvention may be isolated from larger known proteins, such as humanalpha 1 type XVIII collagen and mouse alpha 1 type XVIII collagen,proteins that share a common or similar N-terminal amino acid sequence.Examples of other potential endostatin source materials having similarN-terminal amino acid sequences include Bos taurus pregastric esterase,human alpha 1 type 15 collagen, NAD-dependent formate dehydrogenase (EC1.2.1.2) derived from Pseudomonas sp., s11459 hexon protein of bovineadenovirus type 3, CELF21D12 2 F21d12.3 Caenorhabditis elegans geneproduct, VAL1 TGMV AL1 protein derived from tomato golden mosaic virus,s01730 hexon protein derived from human adenovirus 12, Saccharomycescerevisiae. For example, peptides closely related to endostatin may bederived from BOVMPE 1 pregastric esterase (BOS TAURUS) gene sequencecorresponding to amino acids 502 to 521, and collagen alpha 1 type 15from humans beginning at amino acid 316 ending at 335.

Proteins and peptides derived from these and other sources, includingmanual or automated protein synthesis, may be quickly and easily testedfor endothelial proliferation inhibiting activity using a biologicalactivity assay such as the bovine capillary endothelial cellproliferation assay. Other bioassays for inhibiting activity include thechick CAM assay, the mouse corneal assay, and the effect ofadministering isolated or synthesized proteins on implanted tumors. Thechick CAM assay is described by O'Reilly, et al. in “AngiogenicRegulation of Metastatic Growth” Cell, vol. 79 (2), Oct. 21, 1994, pp.315-328, which is hereby incorporated by reference in its entirety.Briefly, 3 day old chicken embryos with intact yolks are separated fromthe egg and placed in a petri dish. After 3 days of incubation amethylcellulose disc containing the protein to be tested is applied tothe CAM of individual embryos. After 48 hours of incubation, the embryosand CAMs are observed to determine whether endothelial growth has beeninhibited. The mouse corneal assay involves implanting a growthfactor-containing pellet, along with another pellet containing thesuspected endothelial growth inhibitor, in the cornea of a mouse andobserving the pattern of capillaries that are elaborated in the cornea.

Applicants' invention also encompasses nucleic acid sequences thatcorrespond to and code for the endothelial proliferation-inhibitingprotein molecules of the invention, and to monoclonal and polyclonalantibodies that bind specifically to such protein molecules. Thebiologically active protein molecules, nucleic acid sequencescorresponding to the proteins, and antibodies that bind specifically tothe proteins of the present invention are useful for modulatingendothelial processes in vivo, and for diagnosing and treatingendothelial cell-related diseases, for example by gene therapy.

Nucleic acid sequences that correspond to, and code for, endostatin andendostatin analogs can be prepared based upon the knowledge of the aminoacid sequence, and the art recognized correspondence between codons(sequences of three nucleic acid bases), and amino acids. Because of thedegeneracy of the genetic code, wherein the third base in a codon mayvary yet still code for the same amino acid, many different possiblecoding nucleic acid sequences are derivable for any particular proteinor peptide fragment.

Nucleic acid sequences are synthesized using automated systems wellknown in the art. Either the entire sequence may be synthesized or aseries of smaller oligonucleotides are made and subsequently ligatedtogether to yield the full length sequence. Alternatively, the nucleicacid sequence may be derived from a gene bank using oligonucleotidesprobes designed based on the N-terminal amino acid sequence and wellknown techniques for cloning genetic material.

The present invention also includes the detection of endostatin in bodyfluids and tissues for the purpose of diagnosis or prognosis ofangiogenesis-mediated diseases such as cancer. The present inventionalso includes the detection of endostatin binding sites and receptors incells and tissues. The present invention also includes methods oftreating or preventing angiogenic diseases and processes including, butnot limited to, arthritis and tumors by stimulating the production ofendostatin, and/or by administering substantially purified endostatin,or endostatin agonists or antagonists, and/or endostatin antisera orantisera directed against endostatin antisera to a patient. Additionaltreatment methods include administration of endostatin, endostatinfragments, endostatin antisera, or endostatin receptor agonists andantagonists linked to cytotoxic agents. It is to be understood that theendostatin can be animal or human in origin. Endostatin can also beproduced synthetically by chemical reaction or by recombinant techniquesin conjunction with expression systems. Endostatin can also be producedby enzymatically cleaving different molecules, including endostatinprecursors, containing sequence homology or identity with segments ofendostatin to generate peptides having anti-angiogenesis activity.

Passive antibody therapy using antibodies that specifically bindendostatin can be employed to modulate endothelial-dependent processessuch as reproduction, development, and wound healing and tissue repair.In addition, antisera directed to the Fab regions of endostatinantibodies can be administered to block the ability of endogenousendostatin antisera to bind endostatin.

Antibodies specific for endostatin and endostatin analogs are madeaccording to techniques and protocols well known in the art. Theantibodies may be either polyclonal or monoclonal. The antibodies areutilized in well know immunoassay formats, such as competitive andnon-competitive immunoassays, including ELISA, sandwich immunoassays andradioimmunoassays (RIAs), to determine the presence or absence of theendothelial proliferation inhibitors of the present invention in bodyfluids. Examples of body fluids include but are not limited to blood,serum, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterinefluid, saliva, and mucus.

The proteins, nucleic acid sequences and antibodies of the presentinvention are useful for diagnosing and treating endothelialcell-related diseases and disorders. A particularly importantendothelial cell process is angiogenesis, the formation of bloodvessels. Angiogenesis-related diseases may be diagnosed and treatedusing the endothelial cell proliferation inhibiting proteins of thepresent invention. Angiogenesis-related diseases include, but are notlimited to, angiogenesis-dependent cancer, including, for example, solidtumors, blood born tumors such as leukemias, and tumor metastases;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. The endothelial cell proliferation inhibitingproteins of the present invention are useful in the treatment of diseaseof excessive or abnormal stimulation of endothelial cells. Thesediseases include, but are not limited to, intestinal adhesions,atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids.They are also useful in the treatment of diseases that have angiogenesisas a pathologic consequence such as cat scratch disease (Rochele minaliaquintosa) and ulcers (Helobacter pylori).

The endothelial cell proliferation inhibiting proteins can be used as abirth control agent by reducing or preventing uterine vascularizationrequired for embryo implantation. Thus, the present invention providesan effective birth control method when an amount of the inhibitoryprotein sufficient to prevent embryo implantation is administered to afemale. In one aspect of the birth control method, an amount of theinhibiting protein sufficient to block embryo implantation isadministered before or after intercourse and fertilization haveoccurred, thus providing an effective method of birth control, possiblya “morning after” method. While not wanting to be bound by thisstatement, it is believed that inhibition of vascularization of theuterine endometrium interferes with implantation of the blastocyst.Similar inhibition of vascularization of the mucosa of the uterine tubeinterferes with implantation of the blastocyst, preventing occurrence ofa tubal pregnancy. Administration methods may include, but are notlimited to, pills, injections (intravenous, subcutaneous,intramuscular), suppositories, vaginal sponges, vaginal tampons, andintrauterine devices. It is also believed that endostatin administrationwill interfere with normal enhanced vascularization of the placenta, andalso with the development of vessels within a successfully implantedblastocyst and developing embryo and fetus.

Conversely, blockade of endostatin receptors with endostatin analogswhich act as receptor antagonists may promote endothelialization andvascularization. Such effects may be desirable in situations ofinadequate vascularization of the uterine endometrium and associatedinfertilty, wound repair, healing of cuts and incisions, treatment ofvascular problems in diabetics, especially retinal and peripheralvessels, promotion of vascularization in transplanted tissue includingmuscle and skin, promotion of vascularization of cardiac muscleespecially following transplantation of a heart or heart tissue andafter bypass surgery, promotion of vascularization of solid andrelatively avascular tumors for enhanced cytotoxin delivery, andenhancement of blood flow to the nervous system, including but notlimited to the cerebral cortex and spinal cord.

A surprising discovery is that un-refolded and non-soluble recombinantendostatin is also a potent anti-angiogenesis compound which serves as asustained release depot when administered to a patient.

The present invention also relates to methods of using endostatin andendothelial cell proliferation inhibiting peptide fragments ofendostatin, nucleic acid sequences corresponding to endostatin andactive peptide fragments thereof, and antibodies that bind specificallyto endostatin and its peptides, to diagnose endothelial cell-relateddiseases and disorders.

The invention further encompasses a method for identifyingendostatin-specific receptors, and the receptor molecules identified andisolated thereby.

The present invention also provides a method for quantitation ofendostatin receptors.

A particularly important aspect of the present invention is thediscovery of a novel and effective method for treating and curingangiogenesis-dependent cancer in patients. It was unexpectedly foundthat the co-administration of endostatin and angiostatin in an amountsufficient to inhibit tumor growth and cause sustainable regression oftumor mass to microscopic size cures angiogenesis-dependent cancer.Accordingly, the present invention also includes formulations effectivefor treating or curing angiogenesis-dependent cancers and tumors.

More particularly, recombinant mouse endostatin, from insect cells or E.coli, potently inhibits angiogenesis and the growth of metastases andprimary tumors. In a novel method of sustained release, the E.coli-derived recombinant endostatin was administered as an un-refoldedsuspension in an amount sufficient to inhibit angiogenesis, therebyinhibiting tumor growth. Tumor mass was reduced when recombinantendostatin was administered in an amount sufficient to cause regressionof the tumor. Primary tumors of 1-2% of body weight regressed by greaterthan 150-fold to become microscopic dormant lesions when treated byendostatin. Immunohistochemical analysis of the dormant tumors revealedblocked angiogenesis accompanied by high proliferation of the tumorcells balanced by a high rate of tumor cell apoptosis. There was noevidence of toxicity in any of the mice treated with endostatin.

It is contemplated as part of the present invention that endostatin canbe isolated from a body fluid such as blood or urine of patients or theendostatin can be produced by recombinant DNA methods or syntheticpeptide chemical methods that are well known to those of ordinary skillin the art. Protein purification methods are well known in the art and aspecific example of a method for purifying endostatin, and assaying forinhibitor activity is provided in the examples below. Isolation of humanendogenous endostatin is accomplished using similar techniques.

One example of a method of producing endostatin using recombinant DNAtechniques entails the steps of (1) identifying and purifying anendostatin as discussed above, and as more fully described below, (2)determining the N-terminal amino acid sequence of the purifiedinhibitor, (3) synthetically generating a DNA oligonucleotide probe thatcorresponds to the N-terminal amino acid sequence, (4) generating a DNAgene bank from human or other mammalian DNA, (5) probing the gene bankwith the DNA oligonucleotide probe, (6) selecting clones that hybridizeto the oligonucleotide, (7) isolating the inhibitor gene from the clone,(8) inserting the gene into an appropriate vector such as an expressionvector, (9) inserting the gene-containing vector into a microorganism orother expression system capable of expressing the inhibitor gene, and(10) isolating the recombinantly produced inhibitor. The abovetechniques are more fully described in laboratory manuals such as“Molecular Cloning: A Laboratory Manual” Second Edition by Sambrook etal., Cold Spring Harbor Press, 1989.

The gene for endostatin may also be isolated from cells or tissue (suchas tumor cells) that express high levels of endostatin by (1) isolatingmessenger RNA from the tissue, (2) using reverse transcriptase togenerate the corresponding DNA sequence and then (3) using PCR with theappropriate primers to amplify the DNA sequence coding for the activeendostatin amino acid sequence.

Yet another method of producing endostatin, or biologically activefragments thereof, is by peptide synthesis. Once a biologically activefragment of an endostatin is found using the assay system described morefully below, it can be sequenced, for example by automated peptidesequencing methods. Alternatively, once the gene or DNA sequence whichcodes for endostatin is isolated, for example by the methods describedabove, the DNA sequence can be determined, which in turn providesinformation regarding the amino acid sequence. Thus, if the biologicallyactive fragment is generated by specific methods, such as trypticdigests, or if the fragment is N-terminal sequenced, the remaining aminoacid sequence can be determined from the corresponding DNA sequence.

Once the amino acid sequence of the peptide is known, for example theN-terminal 20 amino acids, the fragment can be synthesized by techniqueswell known in the art, as exemplified by “Solid Phase Peptide Synthesis:A Practical Approach” E. Atherton and R. C. Sheppard, IRL Press, OxfordEngland. Similarly, multiple fragments can be synthesized which aresubsequently linked together to form larger fragments. These syntheticpeptide fragments can also be made with amino acid substitutions atspecific locations in order to test for agonistic and antagonisticactivity in vitro and in vivo. Peptide fragments that possess highaffinity binding to tissues can be used to isolate the endostatinreceptor on affinity columns. Isolation and purification of theendostatin receptor is a fundamental step towards elucidating themechanism of action of endostatin. This facilitates development of drugsto modulate the activity of the endostatin receptor, the final pathwayto biological activity. Isolation of the receptor enables theconstruction of nucleotide probes to monitor the location and synthesisof the receptor, using in situ and solution hybridization technology.

Endostatin is effective in treating diseases or processes that aremediated by, or involve, angiogenesis. The present invention includesthe method of treating an angiogenesis mediated disease with aneffective amount of endostatin or endostatin agonists and antagonists.The angiogenesis mediated diseases include, but are not limited to,solid tumors; blood born tumors such as leukemias; tumor metastasis;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic, diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. Endostatin is useful in the treatment of diseaseof excessive or abnormal stimulation of endothelial cells. Thesediseases include, but are not limited to, intestinal adhesions,atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids.Endostatin can be used as a birth control agent by preventingvascularization required for blastocyst implantation and for developmentof the placenta, the blastcyst, the embryo and the fetus.

The synthetic peptide fragments of endostatin have a variety of uses.The peptide that binds to the endostatin receptor with high specificityand avidity is radiolabeled and employed for visualization andquantitation of binding sites using autoradiographic and membranebinding techniques. This application provides important diagnostic andresearch tools. Knowledge of the binding properties of the endostatinreceptor facilitates investigation of the transduction mechanisms linkedto the receptor.

In addition, labeling these peptides with short lived isotopes enablesvisualization of receptor binding sites in vivo using positron emissiontomography or other modern radiographic techniques in order to locatetumors with endostatin binding sites.

Systematic substitution of amino acids within these synthesized peptidesyields high affinity peptide agonists and antagonists to the endostatinreceptor that enhance or diminish endostatin binding to its receptor.Such agonists are used to suppress the growth of micrometastases,thereby limiting the spread of cancer. Antagonists to endostatin areapplied in situations of inadequate vascularization, to block theinhibitory effects of angiostatin and possibly promote angiogenesis.This treatment may have therapeutic effects to promote wound healing indiabetics.

Endostatin peptides are employed to develop affinity columns forisolation of the endostatin receptor from cultured tumor cells.Isolation and purification of the endostatin receptor is followed byamino acid sequencing. Next, nucleotide probes are developed forinsertion into vectors for expression of the receptor. These techniquesare well known to those skilled in the art. Transfection of theendostatin receptor into tumor cells enhances the responsiveness ofthese cells to endogenous or exogenous endostatin and thereby decreasingthe rate of metastatic growth.

Cytotoxic agents, such as ricin, are linked to endostatin, and highaffinity endostatin peptide fragments, thereby providing a tool fordestruction of cells that bind endostatin. These cells may be found inmany locations, including but not limited to, micrometastases andprimary tumors. Peptides linked to cytotoxic agents are infused in amanner designed to maximize delivery to the desired location. Forexample, ricin-linked high affinity endostatin fragments are deliveredthrough a cannula into vessels supplying the target site or directlyinto the target. Such agents are also delivered in a controlled mannerthrough osmotic pumps coupled to infusion cannulae. A combination ofendostatin antagonists may be co-applied with stimulators ofangiogenesis to increase vascularization of tissue. This therapeuticregimen provides an effective means of destroying metastatic cancer.

According to the present invention, endostatin may be used incombination with other compositions and procedures for the treatment ofdiseases. For example, a tumor may be treated conventionally withsurgery, radiation or chemotherapy combined with endostatin and thenendostatin may be subsequently administered to the patient to extend thedormancy of micrometastases and to stabilize any residual primary tumor.

The endostatin of the present invention also can be used to generateantibodies that are specific for the inhibitor. The antibodies can beeither polyclonal antibodies or monoclonal antibodies. These antibodiesthat specifically bind to the endostatin can be used in diagnosticmethods and kits that are well known to those of ordinary skill in theart to detect or quantify the endostatin in a body fluid or tissue.Results from these tests can be used to diagnose or predict theoccurrence or recurrence of a cancer and other angiogenesis mediateddiseases.

The endostatin also can be used in a diagnostic method and kit to detectand quantify antibodies capable of binding endostatin. These kits wouldpermit detection of circulating endostatin antibodies which indicatesthe spread of micrometastases in the presence of endostatin secreted byprimary tumors in situ. Patients that have such circulatinganti-endostatin antibodies may be more likely to develop tumors andcancers, and may be more likely to have recurrences of cancer aftertreatments or periods of remission. The Fab fragments of theseanti-endostatin antibodies may be used as antigens to generateanti-endostatin Fab-fragment antisera which can be used to neutralizethe removal of circulating endostatin by anti-endostatin antibodies.

Another aspect of the present invention is a method of blocking theaction of excess endogenous endostatin. This can be done by passivelyimmunizing a human or animal with antibodies specific for the undesiredendostatin in the system. This treatment can be important in treatingabnormal ovulation, menstruation and placentation, and vasculogenesis.This provides a useful tool to examine the effects of endostatin removalon metastatic processes. The Fab fragment of endostatin antibodiescontains the binding site for endostatin. This fragment is isolated fromendostatin antibodies using techniques known to those skilled in theart. The Fab fragments of endostatin antisera are used as antigens togenerate production of anti-Fab fragment serum. Infusion of thisantiserum against the Fab fragments of endostatin prevents endostatinfrom binding to endostatin antibodies. Therapeutic benefit is obtainedby neutralizing endogenous anti-endostatin antibodies by blocking thebinding of endostatin to the Fab fragments of anti-endostatin. The neteffect of this treatment is to facilitate the ability of endogenouscirculating endostatin to reach target cells, thereby decreasing thespread of metastases.

It is to be understood that the present invention is contemplated toinclude any derivatives of the endostatin that have endothelialinhibitory activity. The present invention includes the entireendostatin protein, derivatives of the endostatin protein andbiologically-active fragments of the endostatin protein. These includeproteins with endostatin activity that have amino acid substitutions orhave sugars or other molecules attached to amino acid functional groups.The present invention also includes genes that code for endostatin andthe endostatin receptor, and to proteins that are expressed by thosegenes.

The proteins and protein fragments with the endostatin activitydescribed above can be provided as isolated and substantially purifiedproteins and protein fragments in pharmaceutically acceptableformulations using formulation methods known to those of ordinary skillin the art. These formulations can be administered by standard routes.In general, the combinations may be administered by the topical,transdermal, intraperitoneal, intracranial, intracerebroventricular,intracerebral, intravaginal, intrauterine, oral, rectal or parenteral(e.g., intravenous, intraspinal, subcutaneous or intramuscular) route.In addition, the endostatin may be incorporated into biodegradablepolymers allowing for sustained release of the compound, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of a tumor or implanted so that the endostatin isslowly released systemically. Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of endostatin throughcannulae to the site of interest, such as directly into a metastaticgrowth or into the vascular supply to that tumor. The biodegradablepolymers and their use are described, for example, in detail in Brem etal., J. Neurosurg. 74:441-446 (1991), which is hereby incorporated byreference in its entirety.

The dosage of the endostatin of the present invention will depend on thedisease state or condition being treated and other clinical factors suchas weight and condition of the human or animal and the route ofadministration of the compound. For treating humans or animals, betweenapproximately 0.5 mg/kilogram to 500 mg/kilogram of the endostatin canbe administered. A more preferable range is 1 mg/kilogram to 100mg/kilogram with the most preferable range being from 2 mg/kilogram to50 mg/kilogram. Depending upon the half-life of the endostatin in theparticular animal or human, the endostatin can be administered betweenseveral times per day to once a week. It is to be understood that thepresent invention has application for both human and veterinary use. Themethods of the present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time.

The endostatin formulations include those suitable for oral, rectal,ophthalmic (including intravitreal or intracameral), nasal, topical(including buccal and sublingual), intrauterine, vaginal or parenteral(including subcutaneous, intraperitoneal, intramuscular, intravenous,intradermal, intracranial, intratracheal, and epidural) administration.The endostatin formulations may conveniently be presented in unit dosageform and may be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and-tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition to the ingredients, particularly mentionedabove, the formulations of the present invention may include otheragents conventional in the art having regard to the type of formulationin question.

Different peptide fragments of the intact endostatin molecule can besynthesized for use in several applications including, but not limitedto the following; as antigens for the development of specific antisera,as agonists and antagonists active at endostatin binding sites, aspeptides to be linked to cytotoxic agents for targeted killing of cellsthat bind endostatin. The amino acid sequences that comprise thesepeptides are selected on the basis of their position on the exteriorregions of the molecule and are accessible for binding to antisera. Theamino and carboxyl termini of endostatin, as well as the mid-region ofthe molecule are represented separately among the fragments to besynthesized. The amino terminus distal to the 20th amino acid andcarboxyl termini of endostatin may contain or be modified to containtyrosine and lysine residues and are labeled with many techniques. Atyrosine or lysine is added to fragments that do not have these residuesto facilitate labeling of reactive amino and hydroxyl groups on thepeptide. These peptide sequences are compared to known sequences usingsequence data banks to determine potential sequence homologies. Thisinformation facilitates elimination of sequences that exhibit a highdegree of sequence homology to other molecules, thereby enhancing thepotential for high specificity in the development of antisera, agonistsand antagonists to endostatin.

Peptides can be synthesized in a standard microchemical facility andpurity checked with HPLC and mass spectrophotometry. Methods of peptidesynthesis, HPLC purification and mass spectrophotometry are commonlyknown to those skilled in these arts.

Peptides and endostatin are also produced in recombinant E. coli, asdescribed below, or in insect or yeast expression systems, and purifiedwith column chromatography.

Endostatin and endostatin derived peptides can be coupled to othermolecules using standard methods. The amino terminus distal to the 20thamino acid and the carboxyl terminus of endostatin may both containtyrosine and lysine residues and are isotopically and nonisotopicallylabeled with many techniques, for example radiolabeling usingconventional techniques (tyrosine residues—chloramine T, iodogen,lactoperoxidase; lysine residues—Bolton-Hunter reagent). These couplingtechniques are well known to those skilled in the art. The couplingtechnique is chosen on the basis of the functional groups available onthe amino acids including, but not limited to amino, sulfhydral,carboxyl, amide, phenol, and imidazole. Various reagents used to effectthese couplings include among others, glutaraldehyde, diazotizedbenzidine, carbodiimide, and p-benzoquinone.

Endostatin peptides are chemically coupled to isotopes, enzymes, carrierproteins, cytotoxic agents, fluorescent molecules and other compoundsfor a variety of applications. The efficiency of the coupling reactionis determined using different techniques appropriate for the specificreaction. For example, radiolabeling of an endostatin peptide or proteinwith ¹²⁵I is accomplished using chloramine T and Na¹²⁵I of high specificactivity. The reaction is terminated with sodium metabisulfite and themixture is desalted on disposable columns. The labeled peptide is elutedfrom the column and fractions are collected. Aliquots are removed fromeach fraction and radioactivity measured in a gamma counter. In thismanner, the unreacted Na¹²⁵I is separated from the labeled endostatinpeptide. The peptide fractions with the highest specific radioactivityare stored for subsequent use such as analysis of the ability to bind toendostatin antisera.

Another application of peptide conjugation is for production ofpolyclonal antisera. For example, endostatin peptides containing lysineresidues are linked to purified bovine serum albumin usingglutaraldehyde. The efficiency of the reaction is determined bymeasuring the incorporation of radiolabeled peptide. Unreactedglutaraldehyde and peptide are separated by dialysis. The conjugate isstored for subsequent use.

Antiserum against endostatin can be generated. After peptide synthesisand purification, both monoclonal and polyclonal antisera are raisedusing established techniques known to those skilled in the art. Forexample, polyclonal antisera may be raised in rabbits, sheep, goats orother animals. Endostatin peptides conjugated to a carrier molecule suchas bovine serum albumin, or endostatin itself, is combined with anadjuvant mixture, emulsified and injected subcutaneously at multiplesites on the back, neck, flanks, and sometimes in the footpads. Boosterinjections are made at regular intervals, such as every 2 to 4 weeks.Blood samples are obtained by venipuncture, for example using themarginal ear veins after dilation, approximately 7 to 10 days after eachinjection. The blood samples are allowed to clot overnight at 4° C. andare centrifuged at approximately 2400×g at 4° C. for about 30 minutes.The serum is removed, aliquoted, and stored at 4° C. for immediate useor at −20 to −90° C. for subsequent analysis.

All serum samples from generation of polyclonal antisera or mediasamples from production of monoclonal antisera are analyzed fordetermination of titer. Titer is established through several means, forexample, using dot blots and density analysis, and also withprecipitation of radiolabeled peptide-antibody complexes using proteinA, secondary antisera, cold ethanol or charcoal-dextran followed byactivity measurement with a gamma counter. The highest titer antiseraare also purified on affinity columns which are commercially available.Endostatin peptides are coupled to the gel in the affinity column.Antiserum samples are passed through the column and anti-endostatinantibodies remain bound to the column. These antibodies are subsequentlyeluted, collected and evaluated for determination of titer andspecificity.

The highest titer endostatin antisera is tested to establish thefollowing; a) optimal antiserum dilution for highest specific binding ofthe antigen and lowest non-specific binding, b) the ability to bindincreasing amounts of endostatin peptide in a standard displacementcurve, c) potential cross-reactivity with related peptides and proteins,including endostatin related species, d) ability to detect endostatinpeptides in extracts of plasma, urine, tissues, and in cell culturemedia.

Kits for measurement of endostatin are also contemplated as part of thepresent invention. Antisera that possess the highest titer andspecificity and can detect endostatin peptides in extracts of plasma,urine, tissues, and in cell culture media are further examined toestablish easy to use kits for rapid, reliable, sensitive, and specificmeasurement and localization of angiostatin. These assay kits includebut are not limited to the following techniques; competitive andnon-competitive assays, radioimmunoassay, bioluminescence andchemiluminescence assays, fluorometric assays, sandwich assays,immunoradiometric assays, dot blots, enzyme linked assays includingELISA, microtiter plates, antibody coated strips or dipsticks for rapidmonitoring of urine or blood, and immunocytochemistry. For each kit therange, sensitivity, precision, reliability, specificity andreproducibility of the assay are established. Intraassay and interassayvariation is established at 20%, 50% and 80% points on the standardcurves of displacement or activity.

One example of an assay kit commonly used in research and in the clinicis a radioimmunoassay (RIA) kit. An endostatin RIA is illustrated below.After successful radioiodination and purification of endostatin or anendostatin peptide, the antiserum possessing the highest titer is addedat several dilutions to tubes containing a relatively constant amount ofradioactivity, such as 10,000 cpm, in a suitable buffer system. Othertubes contain buffer or preimmune serum to determine the non-specificbinding. After incubation at 4 C. for 24 hours, protein A is added andthe tubes are vortexed, incubated at room temperature for 90 minutes,and centrifuged at approximately 2000-2500×g at 4 C. to precipitate thecomplexes of antibody bound to labeled antigen. The supernatant isremoved by aspiration and the radioactivity in the pellets counted in agamma counter. The antiserum dilution that binds approximately 10 to 40%of the labeled peptide after subtraction of the non-specific binding isfurther characterized.

Next, a dilution range (approximately 0.1 pg to 10 ng) of the endostatinpeptide used for development of the antiserum is evaluated by addingknown amounts of the peptide to tubes containing radiolabeled peptideand antiserum. After an additional incubation period, for example, 24 to48 hours, protein A is added and the tubes centrifuged, supernatantremoved and the radioactivity in the pellet counted. The displacement ofthe binding of radiolabeled endostatin peptide by the unlabeledendostatin peptide (standard) provides a standard curve. Severalconcentrations of other endostatin peptide fragments, plasminogen,endostatin from different species, and homologous peptides are added tothe assay tubes to characterize the specificity of the endostatinantiserum.

Extracts of various tissues, including but not limited to, primary andsecondary tumors, Lewis lung carcinoma, cultures of endostatin producingcells, placenta, uterus, and other tissues such as brain, liver, andintestine, are prepared using extraction techniques that have beensuccessfully employed to extract endostatin. After lyophilization orSpeed Vac of the tissue extracts, assay buffer is added and differentaliquots are placed into the RIA tubes. Extracts of known endostatinproducing cells produce displacement curves that are parallel to thestandard curve, whereas extracts of tissues that do not produceendostatin do not displace radiolabeled endostatin from the endostatinantiserum. In addition, extracts of urine, plasma, and cerebrospinalfluid from animals with Lewis lung carcinoma are added to the assaytubes in increasing amounts. Parallel displacement curves indicate theutility of the endostatin assay to measure endostatin in tissues andbody fluids.

Tissue extracts that contain endostatin are additionally characterizedby subjecting aliquots to reverse phase HPLC. Eluate fractions arecollected, dried in Speed Vac, reconstituted in RIA buffer and analyzedin the endostatin RIA. The maximal amount of endostatin immunoreactivityis located in the fractions corresponding to the elution position ofendostatin.

The assay kit provides instructions, antiserum, endostatin or endostatinpeptide, and possibly radiolabeled endostatin and/or reagents forprecipitation of bound endostatin—endostatin antibody complexes. The kitis useful for the measurement-of endostatin in biological fluids andtissue extracts of animals and humans with and without tumors.

Another kit is used for localization of angiostatin in tissues andcells. This endostatin immunohistochemistry kit provides instructions,endostatin antiserum, and possibly blocking serum and secondaryantiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This endostatin immunohistochemistry kit permitslocalization of endostatin in tissue sections and cultured cells usingboth light and electron microscopy. It is used for both research andclinical purposes. For example, tumors are biopsied or collected andtissue sections cut with a microtome to examine sites of endostatinproduction. Such information is useful for diagnostic and possiblytherapeutic purposes in the detection and treatment of cancer.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1

Identification of an Inhibitor of Capillary Endothelial CellProliferation from Hemangioendothelioma Cells

A murine hemangioendothelioma cell line, EOMA (Obeso et al., 1990), wasevaluated for evidence of the production of inhibitors of endothelialcell proliferation. Many of the known endogenous inhibitors ofangiogenesis inhibit the in vitro proliferation of endothelial cells.

Conditioned Media Collection: Cells of the murine hemangioendotheliomacell line EOMA were maintained in DMEM supplemented with 10% bovine calfserum (BCS) and 1% glutamine-penicillin-streptomycin (GPS) in a 37° C.and 10% CO₂ incubator. Conditioned media from EOMA cells (i.e. culturemedia used to grow EOMA cells) was applied to bovine capillaryendothelial cells, stimulated with bFGF, in a 72 hour proliferationassay. The conditioned media reversibly inhibited the proliferation ofcapillary endothelial cells as compared to controls. The pattern ofinhibition was consistent with the presence of inhibitory andstimulatory activity of endothelial cell proliferation (FIG. 1).

EXAMPLE 2

Inhibitory Activity of Endothelial Cell Proliferation is Not Due toAngiostatin

To determine if the inhibitor of capillary endothelial cellproliferation produced by the EOMA cells was angiostatin, pooledconditioned media was applied to a lysine column (lysine conjugated toSepharose™ chromatography beads). Lysine Sepharose binds angiostatin andhas been used for its purification (O'Reilly et al., 1996). Theendothelial cell inhibitory activity was found only in the flow-throughfraction and not in the bound fraction (data not shown). The lack ofbinding of the inhibitory activity to lysine Sepharose suggested thatthe novel inhibitor of endothelial cell proliferation was notangiostatin.

EXAMPLE 3

Purification of a 20 kDa Protein from the Conditioned Media of EOMACells Which Specifically Inhibits Endothelial Cell Proliferation

Because several angiogenesis inhibitors have an affinity for heparin, weapplied the flow-through from the lysine Sepharose column to a heparinSepharose column. The inhibitory activity bound heparin with relativelyhigh affinity and was eluted with 0.6-0.8 M NaCl in 10 mM Tris pH 7.4,as shown in FIG. 2. To further purify the inhibitory activity, thesample was concentrated and applied to a gel filtration (Bio-Rad Bio-GelP-100 fine gel or Pharmacia Sephacryl S-200HR gel) column (see FIG. 3),followed by several cycles of reverse-phase HPLC with a C4 column. Theinhibitory activity was eluted from the C4 column with 40-45%acetonitrile in 0.1% trifluoroacetic acid, as exemplified by FIG. 4.After the final C4 column, the inhibitory activity was associated with aprotein of molecular mass of approximately 20 kDa (reduced) or 18 kDa(non-reduced), by SDS-PAGE, purified to apparent homogeneity.

With respect to Examples 2 and 3, lysine Sepharose, heparin Sepharose,Sephacryl S-200 HR gel (Pharmacia, Uppsala, Sweden), Bio-Gel P-100 finepolyacrylamide gel (Bio-Rad Laboratories, Richmond, Calif.), and aSynChropak RP-4 (100×4.6 mm) C4 reverse-phase column (Synchrom, Inc.,Lafayette, Ind.) were prepared according to the manufacturersrecommendations. A heparin-Sepharose column (50×2.5 cm) was equilibratedwith 50 mM NaCl 10 mM Tris-HCl pH 7.4. Pooled conditioned media wasapplied and the column was washed with the equilibration buffer. Thecolumn was eluted with a continuous gradient of 50 mM-2 M NaCl in 10 mMTris-HCl at pH 7.4 (200 ml total volume) followed by 100 ml of 2 M NaClin 10 mM Tris-HCl at pH 7.4. Fractions were collected and an aliquot ofeach was applied to capillary endothelial cells. Fractions whichinhibited their proliferation were dialyzed (MWCO=6,000-8,000) againstPBS and concentrated using a 4000 MWCO Nanospin concentrator (GelmanSciences, Ann Arbor, Mich.).

A Bio-Gel P-100 column or a Sephacryl S-200 HR column (75×1.5 cm) wasequilibrated with PBS. The sample from heparin Sepharose chromatographywas applied and the column was fluted with the equilibration buffer.Fractions were collected and an aliquot of each was applied toendothelial cells. Fractions which inhibited endothelial proliferationwere concentrated and dialyzed as above.

A SynChropak RPG (100×4.6 mm) column was equilibrated with H₂O/0.1%trifluoroacetic acid (TFA). HPLC-grade reagents (Pierce, Rockford, Ill.)were used. The sample from gel filtration chromatography was applied tothe column and the column was fluted with a gradient of acetonitrile in0.1% TFA at 0.5 ml/minute and fractions were collected. An aliquot ofeach was evaporated by vacuum centrifugation, resuspended in PBS, andapplied to capillary endothelial cells. Inhibitory activity was furtherpurified to apparent homogeneity by at least two subsequent cycles onthe SynChropak C4 column.

To further characterize the 20 kDa inhibitor, we tested it on severalcell lines of endothelial and non-endothelial origin. For the BCE assay,bovine capillary endothelial cells were obtained and grown as previouslydescribed (Folkman et al., 1979). For the proliferation assay, cellswere washed with PBS and dispersed in a 0.05% solution of trypsin. Acell suspension (25,000 cells/ml) was made with DMEM+10% BCS+1% GPS,plated onto gelatinized 24-well culture plates (0.5 mewed), andincubated (37° C., 10% CO₂) for 24 hours. The media was replaced with0.25 ml of DMEM+5% BCS+1% GPS and the test sample applied. After 20minutes of incubation, media and bFGF were added to obtain a finalvolume of 0.5 ml of DMEM+5% BCS+1% GPS+1 ng/ml bFGF. After 72 hours,cells were dispersed in trypsin, resuspended in Hematall (FisherScientific, Pittsburgh, Pa.), and counted by Coulter counter.

Non-endothelial Cell Proliferation Assays

Bovine aortic smooth muscle (SMC), bovine retinal pigment epithelial(RPE), mink lung epithelial (MLE), Lewis lung carcinoma (LLC), and EOMAcells and 3T3 fibroblasts were maintained in a 10% CO2 and 37° C.incubator. For the proliferation assays, cells were washed with PBS andwere dispersed in a 0.05% solution of trypsin. Optimal conditions forthe cell proliferation assays were established for each different celltype. Fetal calf serum (FCS) was used for the RPE, MLE, and LLC cellsand BCS was used for the other cell types. A cell suspension (20,000cells/ml for SMC, RPE, MLE; 15,000 cells/ml for 3T3; 10,000 cells/ml forLLC, EOMA) was made with DMEM+10% bovine serum+1% GPS, plated onto24-well culture plates (0.5 ml/well), and incubated (37° C., 10% CO2)for 24 hours. The media was replaced with 0.5 ml of DMEM+5% bovineserum+1% GPS and the test sample applied. After 72 hours, cells weredispersed in trypsin, resuspended in Hematall (Fisher Scientific,Pittsburgh, Pa.), and counted by Coulter counter.

Only endothelial cells were significantly inhibited, as shown in Table2.

TABLE 2 EFFECT OF ENDOSTATIN ON ENDOTHELIAL AND NON-ENDOTHELIAL CELLPROLIFERATION INHIBITED NON-INHIBITED Bovine capillary Bovine aorticsmooth endothelial cells muscle cells Bovine retinal pigment epithelialcells 3T3 fibroblasts Mink lung epithelial cells EOMAhemangioendothelioma cells Lewis Lung carcinoma cells

The inhibition was first observed at doses of 100 ng/ml with maximalinhibition observed at doses of 600 ng/ml or greater. No significantinhibition was seen for cells of non-endothelial origin at doses 1 logunit higher than those used to inhibit capillary endothelial cellproliferation (data not shown).

EXAMPLE 4

Microsequence Analysis of the 20 kDa Protein Reveals Identity to aFragment of Collagen XVIII

The 20 kDa inhibitor of capillary endothelial cell proliferation fromthe conditioned media was purified to homogeneity, as described in theabove examples, resolved by SDS-PAGE, electroblotted onto PVDF (Bio-Rad,Richmond, Calif.), detected by Ponceau S stain, and excised from themembrane. N-terminal sequence was determined by automated Edmandegradation on an PE/ABD Model 470A protein sequencer (Foster City,Calif.) operated with gas-phase delivery of trifluoracetic acid.

Sequence library searches and alignments were performed against combinedGenBank, Brookhaven Protein, SWISS-PROT, and PIR databases. Searcheswere performed at the National Center for Biotechnology Informationthrough the use of the BLAST network service.

Microsequence analysis of the inhibitor revealed identity to aC-terminal fragment of collagen XVIII. The molecular cloning andsequence of collagen XVIII was first described by Olsen and hiscoworkers and by Rehn and Pihlajaniemi (Oh et al., 1994; Rehn andPihlajaniemi, 1994). Collagen XVIII is a novel collagen which consistsof an N-terminal region with 3 splice variants (Muragaki et al., 1995;Rehn and Pihlajaniemi, 1995), a series of collagen-like domains withinterruptions, and a 35 kDa C-terminal non-collagenous (NC1) domain. An18-amino acid N-terminal microsequence analysis of the purifiedinhibitor of endothelial cell proliferation confirms that it isidentical to a C-terminal fragment of this NC1 domain (FIG. 5). We havenamed this inhibitory fragment of collagen XVIII “endostatin” and it isincluded in the group of molecules that have endostatin activity.

EXAMPLE 5

Recombinant Mouse Endostatin (Baculovirus or E. coli) InhibitsEndothelial Cell Proliferation in Vitro and Angiogenesis in Vivo

The endothelial proliferation cell inhibitor of the present inventioncan be recombinantly expressed in any system used to express proteins.Non-limiting examples of such expressions systems include bacterialexpression systems, yeast expression systems and insect viral expressionsystems.

Recombinant mouse endostatin was expressed using the BacPAK baculovirusexpression system (CLONTECH Laboratories) following the manufacture'sprotocol. Briefly, a cDNA fragment encoding the signal sequence andC-terminal part (endostatin region) of mouse collagen XVIII was insertedinto the pBacPAK8 transfer vector. BacPAK6 viral DNA (expression vector)and plasmid DNA of the pBacPAK8-endostatin clone (modified transfervector) were then cotransfected into insect Sf21 cells and mediacontaining expressed mouse endostatin was collected. The BacPAK6 wasfirst digested with BSU36 enzyme to make it incompetent for independentreplication. The media containing expressed mouse endostatin was appliedto a 1.5×40 cm heparin Sepharose column which had been equilibrated with50 mM NaCl 10 mM Tris pH 7.4. The column was washed with theequilibration buffer and was then eluted sequentially with 0.2 M NaCl,0.4 M NaCl, 0.6 M NaCl, and 1 M NaCl in 10 mM Tris pH 7.4. Allchromatography was performed at 4° C. The 0.6 M NaCl eluant (whichinhibited bovine capillary endothelial cells in a 72 hour proliferationassay) was dialyzed (6-8000 MWCO) against PBS and then reapplied to theheparin Sepharose column. The column was eluted with a gradient of 50 mMNaCl-1.2 M NaCl in 10 mM Tris pH 7.4. An aliquot of each fraction wasapplied to bovine capillary endothelial cells as above and fractionswhich inhibited proliferation were pooled, dialyzed against PBS, andconcentrated using a Nanospin Plus (Gelman Sciences) centrifugalconcentrator (MWCO=10,000). SDS-PAGE of the concentrated sample revealeda discrete band of apparent M_(r) of 20 kDa.

Expression and Purification of Recombinant Mouse Endostatin from E.coli.

The C-terminal part of the cDNA of collagen XVIII was used to amplifythe cDNA of mouse endostatin which was cloned into the pETKH1 vector(pET11d derivative) (Studier et al., 1990). Induction resulted in theproduction of a fusion protein carrying the amino acid sequenceMARRASVGTD SEQ ID NO:2 (RRAS SEQ ID NO:3=protein kinase A recognitionsequence) and 6 histidine residues at the N-terminus followed by thesequence of mouse endostatin (pTBO01#8). The pTB01#8 plasmid wastransformed into BL21:DE3 and the fusion protein was purified onNi⁺²-NTA-beads as described (QiaExpressionist Handbook, Qiagen).Briefly, E. coli were grown until an O.D.₆₀₀ of 0.8-0.9 and expressionof the fusion protein was then induced for 3 hours with 1 mM IPTG. Thebacteria were pelleted and resuspended in 8 M urea, 10 mM Tris-HCl pH8.0 containing 10 mM imidazole and incubated for 1 hour at roomtemperature. The suspension was centrifuged for 15 minutes at 20,000 gand the supernatant incubated with the Ni⁺²-NTA beads for 1 hour at roomtemperature. The suspension was transferred into a column and washedwith 8 M urea, 0.1 M Na-phosphate, 10 mM Tris-HCl pH 6.25 containing 10mM imidazole. The protein was eluted with the same buffer containing 250mM imidazole. The fractions containing endostatin were extensivelydialyzed against PBS. During dialysis, the endostatin precipitated. Theprecipitated endostatin was resuspended in PBS, the proteinconcentration was adjusted to 2-4 mg/ml, and the endostatin was storedat −20° C. until use. For the mouse studies, endostatin was delivered asa suspension in PBS. For the chick chorioallantoic assay, endostatin wasfurther dialyzed against water and then lyophilized.

Recombinant mouse endostatin was produced in both baculovirus and E.coli expression systems. Using sequential heparin Sepharosechromatography, recombinant mouse endostatin was purified to apparenthomogeneity from insect cell media. Ni⁺²-NTA-agarose was used to purifythe E. coli-derived mouse endostatin.

SDS-PAGE revealed a discrete band of approximately 20 kDa orapproximately 22 kDa (reduced) purified to apparent homogeneity forbaculovirus and E. coli-derived recombinant endostatins, respectively(data not shown). Both were dialyzed against PBS prior to use. Afterdialysis, the material from the E. coli system precipitated and wasdelivered as a suspension for subsequent in vivo studies. Recombinantendostatin from baculovirus specifically inhibited the proliferation ofbovine capillary endothelial cells in a dose-dependent fashion. Theinhibition was seen at doses of 100 ng/ml with maximal inhibitionobserved at doses above 600 ng/ml. No significant inhibition ofproliferation of cells of non-endothelial origin or of the EOMA cellswas observed when endostatin was tested at doses up to 1 log unit higherthan those used to inhibit endothelial cell proliferation.

The precipitated (un-refolded) material was not testable in vitro,because of its insolubility. However, a small percentage was soluble inPBS during dialysis and this fraction was used for the endothelial cellassays. Furthermore, after refolding, it was soluble and inhibitedendothelial proliferation (data not shown). When this soluble materialwas applied to endothelial cells, it was found to be inhibitory atconcentrations comparable to both the native and baculovirus-derivedendostatin.

To test for the ability of recombinant mouse endostatin to inhibit invivo angiogenesis, we used the chick chorioallantoic membrane (CAM)assay (Folkman, 1985; Nguyen et al., 1994 which are incorporated hereinby reference). Briefly, three day old fertilized white Leghorn eggs(Spafas, Norwich, Conn.) were cracked, and embryos with intact yolkswere placed in 100×20 mm petri dishes (Folkman, 1985). After 3 days ofincubation (37° C. and 3% CO₂), a methylcellulose (Fisher Scientific,Fair Lawn, N.J.) disc containing endostatin was applied to the CAM ofindividual embryos. The discs were made by desiccation of endostatin in10 μl of 0.45% methylcellulose (in H₂O) on teflon rods. After 48 hoursof incubation, embryos and CAMs were observed by means of astereomicroscope.

At doses of 10-20 μg/10 μl disc, there was potent inhibition of in vivoangiogenesis for both the E. coli and the baculovirus-derivedendostatins in all of the tested CAMs (n=5/group). The E. coliderived-endostatin precipitate gradually dissolved over 5 days andproduced a sustained antiangiogenic effect on the implanted CAMs. Incontrast, the soluble baculovirus-derived endostatin dissolved within 24hours and gave a maximal antiangiogenic effect within a period of 48hours. There was no evidence of toxicity in any of the chick embryostested.

Human Endostatin was produced recombinantly using similar methods.

EXAMPLE 6

Recombinant Mouse Endostatin Inhibits the Growth of Metastases

Because tumor growth is angiogenesis dependent, we treated Lewis lungcarcinoma metastases systematically with recombinant mouse endostatinexpressed in the baculovirus system. Animals with Lewis lung carcinomasof 600-1200 mm³ tumors were sacrificed and the skin overlying the tumorwas cleaned with betadine and ethanol. In a laminar flow hood, tumortissue was excised under aseptic conditions. A suspension of tumor cellsin 0.9% normal saline was made by passage of viable tumor tissue througha sieve and a series of sequentially smaller hypodermic needles ofdiameter 22- to 30-gauge. The final concentration was adjusted to 1×107cells/ml and the suspension was placed on ice. After the site wascleaned with ethanol, the subcutaneous dorsa of mice in the proximalmidline were injected with 1×106 cells in 0.1 ml of saline.

When tumors were 1500 mm³ in size, approximately 14 days after implant,the mice underwent surgical removal of the tumor. The incision wasclosed with simple interrupted sutures. From the day of operation, micereceived daily intraperitoneal injections of recombinant (baculovirus)mouse endostatin or saline. Mice received 0.3 mg/kg/day of endostatinonce daily via subcutaneous injection. When the control mice became sickfrom metastatic disease (i.e., after 13 days of treatment), all micewere sacrificed and autopsied. Lung surface metastases were counted bymeans of a stereomicroscope at 4×magnification.

The growth of Lewis lung carcinoma metastases was almost completelysuppressed by the systemic administration of endostatin at a dose of 0.3mg/kg/day given subcutaneously (7±3 metastases/mouse, n=4, p<0.001). Incontrast, in mice treated with saline after removal of a Lewis lungcarcinoma primary tumor, lung metastases grew rapidly (77±7metastases/mouse). Lung weight, which reflects tumor burden, was 240±25mg in the endostatin treated mice versus 760±30 mg in the control mice(p<0.001). Further, there was no weight loss or evidence of toxicity inany of the mice treated with endostatin.

EXAMPLE 7

Recombinant Mouse Endostatin Inhibits the Growth of Primary Tumors

The yield of endostatin from the baculovirus system was lower than thatof the E. coli system, i.e. 1-2 mg/liter versus 30-40 mg/liter. Wetherefore used E. coli-derived endostatin to study the effect ofendostatin therapy on primary tumor growth. We produced recombinantmouse endostatin from E. coli in sufficient quantity to treat Lewis lungcarcinoma primary tumors. The endostatin was administered as asuspension of the precipitated purified protein to mice bearing Lewislung carcinomas of at least 100-200 mm³. The protein was purified byconventional means but was not refolded prior to its administration tothe mice. The injected precipitate was slowly resorbed over 24-48 hours.

We are unaware of any precedent for the use of an injected depot ofnon-refolded recombinant protein as a sustained-release method inanimals. Nevertheless, endostatin gradually resorbed in vivo and provedto have potent antiangiogenic activity which resulted in prolongedanti-tumor and antiangiogenic activity. Therefore, these data suggest anovel general method for the controlled release of recombinant proteins.Based on this rationale, we have delivered non-refolded recombinantangiostatin from E. coli with similar success.

Accordingly, an aspect of the invention is the administration ofrecombinant endostatin or endostatin analogs in an un-refolded state soas to provide a sustained release depot of endothelial cellproliferation inhibiting protein over a period of at least 8 hours,desirably at least 12 hours, more desirably at least 24 hours or atleast 48 hours, depending on the patient and the disease to be treated.Optionally recombinant and un-refolded angiostatin is administered tosimilarly provide a sustained release depot of protein capable ofreleasing angiostatin protein over a period of at least 8 hours,desirably at least 12 hours, more desirably at least 24 hours or atleast 48 hours, depending on the patient and the disease to be treated.

Mice were implanted with Lewis lung carcinomas as described above.Tumors were measured with a dial-caliper and tumor volumes weredetermined using the formula width²×length×0.52, and the ratio oftreated to control tumor volume (T/C) was determined for the last timepoint. After tumor volume was 100-200 mm³ (0.5-1% of body weight), whichoccurred within 3-7 days, mice were randomized into two groups. Onegroup received recombinant mouse endostatin (E. coli) as a suspension inPBS injected subcutaneously at a site distant from the tumor once daily.The other group received comparable injections of the vehicle alone. Theexperiments were terminated and mice were sacrificed and autopsied whenthe control mice began to die.

The growth of Lewis lung primary tumors was potently suppressed bysystemic therapy with endostatin. Increasing the dose of endostatin wasassociated with improved efficacy (data not shown). At a dose of 10mg/kg, tumor growth was inhibited by 97% as compared to control micetreated with vehicle alone. At a dose of 20 mg/kg given once daily, intwo separate experiments, there was an almost complete regression ofestablished primary tumors (>99% inhibition, p<0.001). These surprisingand unexpected results are shown in FIGS. 6 and 7.

FIGS. 8, 9, 10 and 11 demonstrate the effectiveness of recombinant mouseendostatin for inhibiting tumor growth in a variety of different tumormodels. Also demonstrated is the effectiveness of endostatin derivedfrom human for inhibiting tumor growth.

Immunohistochemical analysis (FIG. 12) of the residual small tumorsshowed a potent inhibition of angiogenesis in the endostatin treatedtumors. Further, the proliferative index of tumors in the endostatin andsaline treated mice was at the same high level in both groups while theapoptotic index increased 8-fold after endostatin therapy. Thus,endostatin therapy results in a similar pattern of tumor dormancy to theone we have previously described for angiostatin (Holmgren et al., 1995;O'Reilly et al., 1996). Further, there was no evidence of drug-relatedtoxicity in any of the treated mice.

After discontinuation of endostatin therapy, a tumor recurred at theprimary site within 5-14 days, became vascularized, and eventuallykilled the mice (data not shown). Notably, we found that E. coli-derivedrecombinant mouse endostatin with a C-terminal polyhistidine tag, whichwas expressed, purified and administered in a comparable fashion to theN-terminal tagged product described above did not inhibit angiogenesisin the CAM assay and had no effect on the growth of Lewis lungcarcinomas (data not shown). These data argue strongly that theanti-tumor and antiangiogenic activity of recombinant endostatin are dueto the specific structure of endostatin and not to a contaminant in thesample.

FIG. 13 shows the results of cycled treatment of Lewis lung carcinomawith recombinant mouse endostatin derived from E. coli. These resultsclearly show reproducible endostatin-dependent regression of tumor mass,followed by tumor growth after termination of endostatin treatment.

These results show that a murine hemangioendothelioma produces a noveland specific 20 kDa inhibitor of endothelial cell proliferation in vitrowhich is also a potent inhibitor of angiogenesis and tumor growth invivo. The N-terminal sequence of this inhibitor, endostatin, isidentical to a C-terminal fragment of collagen XVIII. Systemicadministration of recombinant endostatin potently inhibits angiogenesis,maintains metastases at a microscopic size, and regresses primary tumorsto less than 1 mm³, a reduction of over 150-fold. For as long as miceare treated there is no regrowth of tumors, no evidence of drugresistance, and no toxicity. It is interesting to note that somefragments of the C-terminal domain of collagen type XVIII that arelonger than endostatin do not inhibit endothelial cell proliferation(data not shown).

Endostatin was discovered by the same strategy employed to findangiostatin (O'Reilly et al., 1994), i.e., isolation from a tumor. Whileit is counter-intuitive that tumors should be a source of angiogenesisinhibitors, the results reported here seem to validate this approach.

This leads to the question of why angiogenesis inhibitors should bepresent in tumors that are angiogenic. One possibility is that aninhibitor could be ‘left-over’ after down-regulation of its productionby a tumor cell undergoing the switch to the angiogenic phenotype. Thisappears to be the case for thrombospondin produced by Li-Fraumeni cellsin which the second allele for p53 is mutated or deleted (Dameron etal., 1994).

A second possibility is that the proteolytic activity which accompaniestumor growth, and which is an important component of capillary bloodvessel growth, may also mobilize circulating angiogenesis inhibitorsfrom precursor proteins which are not inhibitory themselves. Angiostatinfor example, inhibits angiogenesis and endothelial cell proliferationwhile plasminogen does not (O'Reilly et al., 1996; O'Reilly et al.,1994). For endostatin, a similar pattern is revealed.

Histology of tumors which regressed under endostatin therapy showedperivascular cuffing of tumor cells surrounding one or more microvesselsin which angiogenesis was blocked. Tumor cells displayed highproliferation balanced by high apoptosis, with no net gain in tumorsize. These data are consistent with a model of a new type of tumordormancy recently proposed (Holmgren et al., 1995). Furthermore,endostatin inhibited proliferation of endothelial cells in vitro, buthad no effect on Lewis lung carcinoma cells, or other cell typesincluding smooth muscle, epithelium, fibroblasts, and the EOMA cell linefrom which it was purified.

The fact that a specific inhibitor of endothelial cell proliferation canregress a tumor to a microscopic size and hold it in a dormant state,despite the fact that the tumor cells are refractory to the inhibitorfrom the outset, indicates that the endothelial population can exertpowerful growth regulatory control over the tumor cells.

The results with endostatin support the theory (Folkman, 1996) that fortherapeutic purposes, it is fruitful to think about a tumor in terms oftwo distinct cell populations: a tumor cell population and anendothelial cell population, each of which can stimulate growth of theother. Growth of each cell population may be optimally inhibited byagents which selectively or specifically target that cell type, i.e.,cytotoxic chemotherapy and antiangiogenic therapy. Furthermore, combinedtreatment of both cell populations may be better than treatment ofeither cell type alone.

To test this theory mice seeded with Lewis lung carcinomas, and bearingtumors which had attained a size of approximately 300 mm³, were treatedwith a combination therapy comprising angiostatin and endostatin, eachat a dose of 20 mg/kg/day for 25 days. Tumors regressed to microscopiclevels by about day 10 of treatment. A completely unexpected finding wasthat tumors remained regressed and dormant for approximately threemonths, even after all treatment was terminated, as is shown in FIG. 14.Experiments of longer duration indicate that an initial treatment oftumor with a combination of angiostatin and endostatin causes a verylong term dormancy, the actual period of which is unknown at this time.

Such long term dormancy is considered a cure to one skilled in the art.For example, the NIH guideline for determining when a treatment iseffective as a cancer cure, is that the tumor remain dormant (i.e. notincreasing in size) for ten times the normal doubling time of the tumor.The dormancy length achieved using a combination of endostatin andangiostatin far exceeds this criteria.

Accordingly, an important aspect of the invention is a compositioncomprising a combination of angiostatin and endostatin, or an endostatinanalog, in amounts sufficient to cause long term dormancy, or cure, ofangiogenesis-dependent cancers when administered to patients withangiogenesis-dependent cancers. Administration can be systemically, forexample by injection, in which case the dosage is determined dependingupon the patient and the particular cancer, but which generally is atleast 0.2 mg/kg/day, desirably at least 2.0 mg/kg/day, more desirably atleast 20 mg/kg/day. Generally, the composition is administered daily forat least 10 days, desirably at least 20 days, more desirably at least 25days. Alternative systemic administration routes include, orally wherethe composition is formulated, for example into coated microbeads, toprotect the protein from inactivating digestive environments;transdermally; and via pump.

Alternatively, different dosages and treatment periods can be used ifthe composition is administered locally to an angiogenesis-dependentsite, such as a tumor. Such administration may be, for example, surgicalimplantation or local injection into, or near by, the site.

EXAMPLE 8

Isolation of the Putative Receptor for Endostatin

Both endostatin and angiostatin appear to be specific inhibitors ofendothelial cell proliferation. Therefore, it is likely that endostatinbinds to specific structures exclusively expressed on the surface ofendothelial cells. We are not aware of the existence of any otherspecific inhibitors of endothelial cell proliferation.

Identifying and isolating proteins which specifically bind to endostatinis accompanied by methods well known in the art, for example by affinitychromatography and expression cloning.

Affinity chromatography. Bovine Capillary Endothelial cells (BCE) areradiolabeled with [35S]-methionine, total cell and membrane extractsprepared and applied to affinity columns prepared with endostatin. As anegative control, fibroblast protein extracts are isolated in a similarway. Bound proteins are eluted from the column using a NaCl gradient andthe different fractions are analyzed using standard SDS-PAGE andautoradiography. This procedure yields proteins that are tightly boundto the endostatin column and present only in the endothelial cellderived fractions. Comparing the gel electrophoretic patterns of the twocell types reveals expressed proteins unique to the BCE cells. Proteinsequences subsequently are determined and corresponding gene(s) cloned.A cDNA library of bovine capillary endothelial cells, is prepared andscreened with a degenerative oligo based PCR technique to locate thecDNA(s) of the endostatin-specific binding protein(s). Hybridizationusing degenerative oligonucleotides to the corresponding cDNA, is alsoused to identify genes of endostatin binding proteins. Another approachis to raise antibodies against the peptide sequences with methodsdescribed earlier in the Detailed Description and immunoscreen the samelibrary.

Expression cloning. A cDNA library of BCE cells is prepared. Poly-A mRNAis isolated from BCE cells whose proliferation has previously beeninhibited by endostatin. These cells express an endostatin bindingprotein. The corresponding cDNA library is transfected into cellsallowing high expression of the various cDNAs. Binding activity ofendostatin to cells which express the receptor protein on the surface isused as a positive selection of these cells. To select for these cells,purified endostatin is labeled with biotin and consequently detectedusing either streptavidin coupled magnetic-beads or FACS sorting.Alternatively, an antibody against endostatin is used for screening.After selection of the positive cells, the corresponding plasmids areisolated, amplified and transfected again into high expression cells.After several rounds of positive selection, plasmids are analyzed foridentical inserts using endonuclease digestion and PCR. Using thesedata, complementation groups are formed, sequenced and analyzed with theBLAST network program. In addition to computer analysis, individualcDNAs are re-transfected into high expression cells and tested forendostatin binding activity under different conditions (e.g.,competition with non-labeled endostatin, time-course of binding,Scatchard analysis, etc. in other words the use of “classical” receptorcharacterization procedures known to those skilled in the art).

EXAMPLE 9

Determination of the Minimal Region of the Mouse Endostatin ProteinResponsible for its Antiangiogenic Activity

Different PCR primers are designed, the corresponding cDNAs cloned intothe E. coli expression system, and the different endostatin fragmentspurified to homogeneity. The full length cDNA is cut from both the N-and C-terminus. As a first screen the capillary endothelialproliferation assay and the chick embryo assay are used to determine theresidual activity compared to the full length fragment.

EXAMPLE 10

Determination of the Putative Enzyme(s) which may Release Endostatinfrom Collagen XVIII

Collagen XVIII belongs to the non-fibrillar collagen type family and canbe found in three different splicing variants encoding for proteins with1315-, 1527-, and 1774 amino acid residues (Rehn, PNAS 91:4234, 1994).The difference is caused by alterations in the N-terminal part of thegene and therefore all three splicing variants could potentially be thesource of endostatin which itself is a fragment of the non-collagenousdomain 11 (NC11). The function of collagen XVIII is not known, butbecause its message is substantially expressed in highly vascularizedorgans, a role in perivascular matrix assembly and/or structure has beenproposed (Oh, et al., Genomics, 19:494, 1994). A first clue about thefunction of collagen XVIII came from the purification of endostatin as apotent inhibitor of endothelial cell proliferation.

From this preliminary data and from our initial observation thatendostatin was purified from conditioned medium of ahemangioendothelioma (EOMA), we asked whether the enzyme(s) whichrelease endostatin from collagen XVII could be identified.

The last 325 amino acid residues, encoding for the NC11 domain, areexpressed in E. coli and the insect cell baculovirus system, thepurified protein is used as a substrate to identify enzymes that clonethis region of collagen XVIII. By PCR, a cDNA fragment encoding the NC11domain is cloned into an E. coli expression vector (pET series) whichallows high expression of the target protein after induction with IPTG.Alternatively, a vector suitable for insect cell expression is used. Theproteins are tagged with the HIS₆-Tag located on the C-terminus forpurification using Ni²⁺-NTA-beads. An Ni²⁺-NTA-alkaline phosphataseconjugate can detect the C-terminus by Western blotting. Anotherconstruct is made which not only has a HIS₆-Tag on the C-terminus, butwill also encode the hemagglutinin (HA-tag on the N-terminus. This isdetected by Western blotting with an HA-specific monoclonal antibody.The N- and C-terminus of the protein followed after incubation with EOMAsupernatant and different metalloproteinase extracts.

Cleavage product is detected by SDS-PAGE analysis or Western blotting,the protein is re-purified using the Ni²⁺-NTA beads, eluted withimidazole, dialyzed against PBS and tested for inhibitor activity in thevarious in vitro and in vivo assays (e.g., endothelial cellproliferation, chick embryo, and mouse corneal assay). If the purifiedcleavage product shows inhibitory activity, N-terminal amino acidsequencing is performed and compared to the original starting sequenceof endostatin obtained from the EOMA supernatant. Accordingly, thecleavage procedure can be scaled up to purify sufficient protein fortesting in tumor-bearing mice, and to compare this activity to that ofthe full length NC11 domain.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 3 <210> SEQ ID NO 1 <211> LENGTH: 20<212> TYPE: PRT <213> ORGANISM: Murinae gen. sp. <400> SEQUENCE: 1His Thr His Gln Asp Phe Gln Pro Val Leu Hi #s Leu Val Ala Leu Asn  1               5  #                 10  #                 15Thr Pro Leu Ser              20 <210> SEQ ID NO 2 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: vector      pETKH1 <400> SEQUENCE: 2 Met Ala Arg Arg Ala Ser Val Gly Thr Asp  1               5  #                 10 <210> SEQ ID NO 3<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: vector       pETKH1 <400> SEQUENCE: 3 Arg Arg Ala Ser   1

What is claimed is:
 1. An endostatin protein consisting of a fragment ofa NC1 region of a collagen protein, wherein the fragment inhibitsangiogenesis, the endostatin protein having a molecular weight ofapproximately 18 kDa as determined by non-reducing gel electrophoresis,wherein the N-terminal amino acid sequence is shown in SEQ ID NO:1 andwherein the protein is further characterized by its ability tospecifically inhibit proliferating cultured endothelial cells.
 2. Theprotein of claim 1, wherein the protein is a fragment of a non-fibrillarcollagen protein.
 3. The protein of claim 1, wherein the protein is afragment of a collagen type XVIII protein.
 4. The protein of claim 1,wherein the protein is a fragment of a collagen type XV protein.
 5. Theprotein of claim 1, wherein the protein is produced recombinantly. 6.The protein of claim 1, wherein the protein is naturally occurring. 7.The protein of claim 1, wherein the protein is human.
 8. The protein ofclaim 1, wherein the protein inhibits angiogenesis in vivo.
 9. Theprotein of claim 1, wherein the protein inhibits angiogenesis in vitro.10. A composition comprising, an endostatin protein combined with anangiostatin protein, wherein the endostatin protein consists of afragment of a NC1 region of a collagen protein, wherein the angiostatinprotein is a fragment of a kringle region of plasminogen and wherein theendostatin protein and the angiostatin protein are further characterizedby their ability to inhibit angiogenesis, the endostatin protein havinga molecular weight of approximately 18 kDa as determined by non-reducinggel electrophoresis, wherein the N-terminal amino acid sequence is shownin SEQ ID NO:1 and wherein the protein is further characterized by itsability to specifically inhibit proliferating cultured endothelialcells.
 11. The composition of claim 10, wherein the endostatin proteinis a fragment of a non-fibrillar collagen protein.
 12. The compositionof claim 10, wherein the endostatin protein is a fragment of a collagentype XVIII protein.
 13. The composition of claim 10, wherein theendostatin protein is a fragment of a collagen type XV protein.
 14. Thecomposition of claim 10, wherein the endostatin protein and angiostatinprotein are produced recombinantly.
 15. The composition of claim 10,wherein the endostatin protein and angiostatin protein are naturallyoccurring.
 16. The composition of claim 10, wherein the endostatinprotein and angiostatin protein are human.
 17. The composition of claim10, wherein the endostatin protein and angiostatin protein inhibitangiogenesis in vivo.
 18. The composition of claim 10, wherein theendostatin protein and angiostatin protein inhibit angiogenesis invitro.